PROJECT SUMMARY Inflammatory bowel disease (IBD) affects approximately 3.2 million individuals in the US and several million worldwide. One of the major discoveries in the last 20 years in IBD research has been the key role cytokines play in intestinal inflammation and the development of anti-cytokine treatments for IBD patients, including anti- TNF? and anti-IL23 therapies. In spite of these important advances, much more needs to be learned concerning the basic mechanisms by which cytokines regulate normal gut homeostasis and mediate chronic intestinal inflammation and IBD. The present competitive renewal application will focus on the TNF superfamily member TL1A and its receptor DR3, for which strong evidence suggests a major role in the pathogenesis of human and experimental IBD. The central hypothesis of this proposal is that TL1A/DR3 is a master regulator of chronic intestinal inflammation in IBD. Based on this concept, targeted manipulation of TL1A/DR3 may provide a new therapeutic approach for the treatment of this devastating disease. The following three specific aims are designed to continue our ongoing efforts to understand the role of cytokines in mediating intestinal inflammation with a particular focus on TL1A. 1) Determine the cellular mechanisms of TL1A/DR3 signaling in experimental CD-like ileitis. We will test the hypothesis that TL1A/DR3 signaling regulates the balance between effector and regulatory T cells by using models of DR3 deletion and DR3 activation (SAMPDR3-/- and SAMP treated with a DR3 stimulating antibody, i.e. ?DR3-SAMP) and we will determine how TL1A/DR3 regulates the frequency and function of Th1, Th2, Th9, and Tregs in these models. 2) Evaluate the role of TL1A/DR3 in regulating the balance between pathogenic and protective ILCs in experimental CD-like ileitis. We will determine the effects of DR3 stimulation or deletion on lamina propria (LP) ILCs in SAMP mice, as well as characterize ILCs in GF-SAMPDR3-/-. We will also evaluate the functional effects of ILC3s by performing reconstitution of specific ILC3 subsets and determine whether their effects on ileitis and epithelial barrier function in SAMP mice. 3) Investigate the effects of TL1A/DR3 signaling on the composition and function of the gut microbiome in experimental CD-like ileitis. We will first determine the composition of stool content and intestinal mucosa of SAMPDR3-/- and ?DR3-SAMP littermates by state-of-the-art 16S rRNA and multi-kingdom metagenomics, and determine differences with control mice. We will then perform a series of in vivo experiments using fecal material transplantation (FMT) from these mice into SAMP and AKR control germ-free (GF) recipients. Finally, we will perform FMT from CD patients carrying TL1A mutations into GF SAMP recipients to determine whether the same effects observed using mouse flora are also present with human flora. The overall objective of this proposal is to understand basic mechanisms of TL1A/DR3-driven intestinal inflammation and its relationship with the gut microbiome in order to develop novel treatment modalities for patients with IBD.